The present invention relates to a novel polypeptide, a novel protein comprising a homologous dimer or a heterologous dimer of said polypeptide, a novel DNA coding for said peptide, a recombinant DNA molecule comprising said DNA, a transformant which is transformed with said recombinant DNA molecule, an antibody against said novel polypeptide or said novel protein, a process for preparing said novel polypeptide or said novel protein, and an agent for treating feline viral diseases comprising said novel protein or said novel antibody. More particularly, the present invention relates to a feline cytokine protein comprising two distinct novel polypeptides having an activity to damage virus-infected cells by activating feline cytotoxic T lymphocytes and a gene coding for said cytokine protein as well as a process for preparing said feline cytokine protein.
A cat is such an animal that has been loved by humans as a pet from ancient times and, in modern times, called as Companion species, is becoming a member of a human society. On the other hand, a cat has hitherto greatly contributed to humans as an experimental animal in various fields such as medicine, pharmaceutics, animal husbandry veterinary and psychology, and in recent years, the contribution of a cat has further increased to be used in an effectiveness assay or safety test for drugs. With increasing social significance of a cat, there is a high interest in feline diseases, especially feline infectious diseases, and thus more efficacious method for treating these diseases is desired.
Many feline viral diseases, attack of which often leads to serious conditions, are known. For example, an upper tracheal disease caused by feline herpes virus type 1 (FHV-1) or feline calicivirus (FCV) is acute and highly lethal. In addition to this, diseases caused by feline immunodeficiency virus, feline infectious peritonitis virus, feline parvovirus, etc. are also highly lethal and have been great concern. Although some prophylactic vaccines have been developed for these viral diseases, many of these vaccines are not fully efficacious due to diversity of viral serotype. Furthermore, once a cat is infected with virus and after the onset of viral diseases, vaccines are not substantially efficacious any more, and hence, protection from secondary bacterial infections with antibiotics, sulfonamides etc. or symptomatic treatment with vitamins or nutrients have primarily been carried out. That is, presently no efficacious medicaments are available for treating the viral diseases.
Host immunity to microorganisms including viruses consists of a humoral immunity by an antibody and a cellular immunity by a lymphocyte. A cellular immunity reaches at its maximum level seven to ten days after infection and thereafter declines rapidly. On the other hand, antibody production starts to increase a week after infection, reaches at its maximum level around three to four weeks after infection and thereafter declines slowly. Since a neutralizing antibody is effective at the early stage of viral infection as well as passive immunity, the crux of developing a vaccine for prophylaxis of diseases is to produce a neutralizing antibody in any way. Once infection has been established, however, a neutralizing antibody does not usually function effectively with the lapse of time after infection. Moreover, a neutralizing antibody is not so effective in case of a persistent infection (e.g. herpes virus) wherein a virus remains within cells and infects through cells to cells or in case that a virus is likely to mutate even after infection to exhibit resistance against a neutralizing antibody (e.g. immunodeficiency virus). In such cases, it is generally believed that only a cellular immunity is effective which is mediated by cytotoxic T lymphocytes (CTL) which find out and eliminate those cells infected with virus. In general, a cytotoxic activity declines as symptoms became serious with the lapse of time after infection, resulting in more serious symptoms. In this context, if cytotoxic T lymphocytes could be activated, then the progress of disease can be retarded and rapid recovery will be expected (A. Capron et al., Current Topics in Microbiology and Immunology 189 Springer-Verlag, 1994).
There is a human therapy by activation of cytotoxic T lymphocytes wherein cytotoxic T lymphocytes are recovered from peripheral blood and, after nonspecific activation in vitro, reintroduced into the living body. In case of cats, however, this therapy is not applicable since sufficient amount of cytotoxic T lymphocytes cannot be recovered from peripheral blood due to a small body size of cats. That is, at present, there is not found any therapy for effective treatment of feline viral infectious diseases through activation of cytotoxic T lymphocytes. Although significance of a cellular immunity for treating viral infectious diseases is suggested, there has not hitherto been reported a cellular immunity-based medicament for feline viral infectious diseases, especially a medicament for activating cytotoxic T lymphocytes.
A cytokine is possibly involved in activation of cytotoxic T lymphocytes. Feline cytokines reported hitherto include, for example, erythropoietin (D. Wen et al., Blood Vol. 82, 1507-1516, 1993), a interferon (J. T. Yamamoto et al., Vet. Immunol. Immunopathol. Vol. 11, 1-19, 1986), and the like. However, there has hitherto been no report that any of these cytokines activated cytotoxic T lymphocytes. On the other hand, human or mouse interleukin 12 (hereinafter referred to as xe2x80x9cIL12xe2x80x9d) is known which has an ability to activate T lymphocytes or to induce y interferon and activates cytotoxic T lymphocytes. Human and mouse IL12s cloned hitherto are known to exhibit the activity with a heterologous dimer consisting of an alpha chain (p35) and a beta chain (p40) (Annu. Rev. Immunol. Vol. 13, 251-276, 1995). It is also reported that human and mouse IL12 s show species specificity (S. F. Wolf et al., J. Immunol. Vol. 136, 3074-3081, 1991). As to feline IL12, a partial amino acid sequence of p35 has been reported (K. Bush et al., Molecular Immunology, Vol. 31, 1373-1374, 1994) with no disclosure of a biological activity of IL12 while an amino acid sequence of p40 has not yet been reported. Accordingly, at present, there is no substantial report as to feline cytokines which activate cytotoxic T lymphocytes.
Under the circumstances, the present inventors have thoroughly studied in order to find out feline cytokines which activate cytotoxic T lymphocytes, and as a result, have found a proteinaceous agent (novel feline cytokine) which enhances a cytotoxic activity of feline lymphocytes (especially cytotoxic T lymphocytes: CTL) in a culture supernatant of activated feline splenocytes. Furthermore, the present inventors have isolated and purified the novel feline cytokine protein from the recovered culture supernatant, elucidated their properties, expressed them in an animal cell with a gene engineering technique, and found that the resulting novel feline cytokine protein had an activity to enhance the cytotoxic activity of feline cytotoxic T lymphocytes, thereby completing the present invention.
That is, an object of the present invention is to provide a novel feline cytokine protein which enhances a cytotoxic activity of feline cytotoxic T lymphocytes as well as a polypeptide comprising a partial amino acid sequence of said novel feline cytokine protein.
Another object of the present invention is to provide a gene coding for a novel feline cytokine protein which enhances a cytotoxic activity of feline cytotoxic T lymphocytes or a polypeptide comprising a partial amino acid sequence of said novel feline cytokine protein as well as a recombinant DNA molecule for expressing said gene.
Further object of the present invention is to provide a process for preparing a novel feline cytokine protein which enhances a cytotoxic activity of feline cytotoxic T lymphocytes and a polypeptide comprising a partial amino acid sequence of said novel feline cytokine protein from microorganisms or animal cells transformed with said recombinant DNA molecule.
Still further object of the present invention is to provide a monoclonal antibody and a polyclonal antibody produced by using as an immunogen the thus obtained novel feline cytokine protein or the polypeptide comprising a partial amino acid sequence of said novel feline cytokine protein.
Still another object of the present invention is to provide an agent for treating feline viral infectious diseases comprising as an active ingredient the novel feline cytokine protein which enhances a cytotoxic activity of feline cytotoxic T lymphocytes.
The novel feline cytokine proteins as found out by the present inventors are a protein with a molecular weight of about 40,000 having the amino acid sequence as shown in Sequence Listing, SEQ ID NO: 1 or SEQ ID NO:2 (hereinafter also referred to as xe2x80x9cFLAF p40xe2x80x9d), and a protein with a molecular weight of about 35,000 having the amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4 (hereinafter also referred to as xe2x80x9cFLAF p35xe2x80x9d). The present invention also encompasses a peptide comprising a partial amino acid sequence of these proteins.
The present invention also encompasses a polyclonal antibody and a monoclonal antibody prepared by using as an immunogen the novel feline cytokine protein or the peptide having a partial amino acid sequence of said protein. Furthermore, the present invention encompasses an antibody obtained by introducing an expression vector wherein a gene coding for the polypeptide or a part of said gene is incorporated into an animal body in a conventional manner for expression within said animal body.
The present invention also encompasses gene fragments which code for the novel feline cytokine protein and have the nucleotide sequence as shown in SEQ ID NO: 5, 6 or 7 for FLAF p40 or the nucleotide sequence as shown in SEQ ID NO: 8 or 9 for FLAF p35, a gene fragment coding for a peptide comprising a partial amino acid sequence of said protein, as well as a recombinant DNA molecule comprising these gene fragments.
In addition, the present invention encompasses a transformant (E. coli, yeast, an insect cell, an animal cell, a plant cell) transformed with an expression vector such as a plasmid comprising the recombinant DNA molecule. The present invention also encompasses a process for preparing a desired novel feline cytokine protein or a peptide comprising a partial amino acid sequence of said protein by using said transformant.